The present invention is generally in the area of cloning, expression, and regulation of an endothelial cell protein C/activated protein C receptor.
Protein C plays a major role in the regulation of blood coagulation. Patients deficient in protein C usually exhibit life threatening thrombotic-complications in infancy (Seligsohn et al., (1984) N. Engl. J. Med. 310, 559-562; Esmon, (1992) Trends Cardiovasc. Med. 2, 214-220) that are corrected by protein C administration (Dreyfus et al., (1991) N. Engl. J. Med. 325, 1565-1568). In addition, activated protein C (APC) can prevent the lethal effects of E. coli in baboon models of gram negative sepsis (Taylor et al., (1987) J. Clin. Invest. 79; U.S. Pat. No. 5,009,889 to Taylor and Esmon) and preliminary clinical results suggest that protein C is effective in treating certain forms of human septic shock (Gerson et al., (1993) Pediatrics 91, 418-422). These results suggest that protein C may both control coagulation and influence inflammation. Indeed, inhibition of protein S, an important component of the protein C pathway, exacerbates the response of primates to sublethal levels of E. coli and augments the appearance of TNF in the circulation (Taylor et al., (1991) Blood 78, 357-363). The mechanisms involved in controlling the inflammatory response remain unknown.
Protein C is activated when thrombin, the terminal enzyme of the coagulation system, binds to an endothelial cell surface protein, thrombomodulin (Esmon, (1989) J. Biol. Chem. 264, 4743-4746; Dittman and Majerus, (1990) Blood 75, 329-336; Dittman, (1991) Trends Cardiovasc. Med. 1, 331-336). In cell culture, thrombomodulin transcription is blocked by exposure of endothelial cells to tumor necrosis factor (TNF) (Conway and Rosenberg, (1988) Mol. Cell. Biol. 8, 5588-5592) and thrombomodulin activity and antigen are subsequently internalized and degraded (Lentz et al., (1991) Blood 77, 543-550, Moore,K. L., et.al., (1989) Blood 73, 159-165). In addition, C4bBP, a.regulatory protein of the complement system, binds protein S to form a complex that is functionally inactive in supporting APC anticoagulant activity in vitro (Dahlbxc3xa4ck, (1986) J. Biol. Chem. 261, 12022-12027) and in vivo (Taylor,et al., 1991). Furthermore, C4bBP behaves as an acute phase reactant (Dahlbxc3xa4ck, (1991) Thromb. Haemostas. 66, 49-61). Thus, proteins of this pathway not only appear to regulate inflammation, but they also interact with components that regulate inflammation, and they themselves are subject to down regulation by inflammatory mediators.
Given the central role of the protein C pathway in regulating the host response to inflammation and the critical role of the pathway in controlling blood coagulation, it is important to identify and characterize all of the components that interact with the system. This is especially true since the molecular basis of the anti-inflammatory effects of the protein C pathway components have yet to be elucidated at the molecular level.
It is therefore an object of the present invention to provide a cellular receptor for protein C and activated protein C.
It is a further object of the present invention to provide nucleotide sequences encoding the cellular receptor and amino acid characterization of the receptor which allows expression of recombinant native and modified forms of the receptor.
It is another object of the present invention to provide methods of modulating the inflammatory response involving protein C and activated protein C.
An endothelial cell protein C binding protein (referred to herein as xe2x80x9cEPCRxe2x80x9d) has been cloned and characterized. The protein is predicted to consist of 238 amino acids, which includes a 15 amino acid signal sequence at the N-terminus, and a 23 amino acid transmembrane region which characterizes the receptor as a type 1 transmembrane protein. The protein binds with high affinity to both protein C and activated protein C (Kd=30 nM) and is calcium dependent. The message and binding function of the receptor are both down regulated by cytokines such as TNF.
These results identify a new member of a complex pathway that, like other members of the pathway, is subject to regulation by inflammatory cytokines, and can therefore be used to modulate inflammatory reactions in which protein C or activated protein C is involved. Inhibition of the inflammatory response can be obtained by infusing soluble EPCR. Alternatively, localizing EPCR to surfaces in contact with blood will render the surfaces anticoagulant by virtue of the ability of EPCR to bind and concentrate the anticoagulant activated protein C at the surface. Alternatively, the function of EPCR can be enhanced by overexpressing the EPCR in endothelium that could be used to coat vascular grafts in patients with vascular disease or on stents in cardiac patients.